Need to grow in the absence of CO2 - (Oct/01/2009 )
Wow..this is such a spot on for my problems. Thanks Alinor19 for posting up this Q. I am trying to set up a tissue culture project but due to some problems we are not able to have a CO2 incubator (well at least for few months i hope so..)
I have already heard about the CO2 independent medium (Gibco) and the Leibovitz L-15 media but the literatures that i found usually just used it to prepare their cells for thier particular experiments so that their cells will still be happy during the course of the experiments out of the CO2 incubator. But they still grow and maintained their cells in other medias (DMEM etc) in Co2 incubator.
So, base on the replies given in this discussions, does it means that i can grow and maintain my cells using either CO2 independent medium or L15 or add HEPES buffer in other medias in a normal incubator (without CO2 supply)?
Thanx!
newbie101 on Oct 8 2009, 04:41 AM said:
I have already heard about the CO2 independent medium (Gibco) and the Leibovitz L-15 media but the literatures that i found usually just used it to prepare their cells for thier particular experiments so that their cells will still be happy during the course of the experiments out of the CO2 incubator. But they still grow and maintained their cells in other medias (DMEM etc) in Co2 incubator.
So, base on the replies given in this discussions, does it means that i can grow and maintain my cells using either CO2 independent medium or L15 or add HEPES buffer in other medias in a normal incubator (without CO2 supply)?
Thanx!
Yes As far as i am aware i think this should work fine,
adding 25mM HEPES to most medias including DMEM/F12 or DMEM buffers them like CO2 does in a CO2
incubator
Cotchy.
KaurJ on Oct 7 2009, 04:52 PM said:
to: Rhombus - why do the cells have to be sealed from environment when using HEPES?
I am currently trying to use HEPES to grow cells in 96 well microtiter plates and after removing cells from CO2 environment the medium turns alkaline (phenol red goes purple), since I did not seal the plate I was wondering whether this might be the reason. I couldn't find any mention of the need to seal the cells in literature.
i don think Rhombus means actually sealing the flask, i think you just just use a normal culture flask - without a vent at the top, and keep the lid closed, this ensures a contant internal environment
Rhombus correct me if i am wrong.
I used to use flasks without filters in a CO2 incubator, we would slightly loosen the cap. Would you have a loosened cap with flasks outside or completely sealed?
This is a good topic as it is something I have a problem with too. One of my incubators has broken so I am left with only one CO2 incubator which isn't enough.
I am using CHO freestyle cells from invitrogen. However my collegue tried using HEPES but got really bad cell clumping and no cell growth. Conditions as follows:
25mM Hepes in the Freestyle CHO medium, shaking at 125rpm at 37 C in a 250 ml flask
These cells normally need to be at 8% CO2 and are unusually in suspension. I don't know if this makes a difference, as most of our cell lines are adherent and at 5% CO2.
Should I just try a lower concentration? Or could I use NaHCO3? (I have read about the use of that but have no clue how to do it)